We have developed a three-dimensional (3D) bioprinting system capable of multimaterial and multiscale deposition to enable the next generation of “bottom-up” tissue engineering. This area of research resides at the interface of engineering and life sciences. As such, it entails the design and implementation of diverse elements: a novel hydrogel-based bioink, a 3D bioprinter, automation software, and mammalian cell culture. Our bioprinter has three components uniquely combined into a comprehensive tool: syringe pumps connected to a selector valve that allow precise application of up to five different materials with varying viscosities and chemistries, a high velocity/high-precision x–y–z stage to accommodate the most rapid speeds allowable by the printed materials, and temperature control of the bioink reservoirs, lines, and printing environment. Our custom-designed bioprinter is able to print multiple materials (or multiple cell types in the same material) concurrently with various feature sizes (100 μm–1 mm wide; 100 μm–1 cm high). One of these materials is a biocompatible, printable bioink that has been used to test for cell survival within the hydrogel following printing. Hand-printed (HP) controls show that our bioprinter does not adversely affect the viability of the printed cells. Here, we report the design and build of the 3D bioprinter, the optimization of the bioink, and the stability and viability of our printed constructs.

References

References
1.
Chang
,
C. C.
,
Boland
,
E. D.
,
Williams
,
S. K.
, and
Hoying
,
J. B.
,
2011
, “
Direct-Write Bioprinting Three-Dimensional Biohybrid Systems for Future Regenerative Therapies
,”
J. Biomed. Mater. Res. Part B Appl. Biomater.
,
98B
(
1
), pp.
160
170
.
2.
Petersen
,
T. H.
,
Calle
,
E. A.
,
Zhao
,
L.
,
Lee
,
E. J.
,
Gui
,
L.
,
Raredon
,
M. B.
,
Gavrilov
,
K.
,
Yi
,
T.
,
Zhuang
,
Z. W.
,
Breuer
,
C.
,
Herzog
,
E.
, and
Niklason
,
L. E.
,
2010
, “
Tissue-Engineered Lungs for in vivo Implantation
,”
Science
,
329
(
5991
), pp.
538
541
.
3.
Uygun
,
B. E.
,
Soto-Gutierrez
,
A.
,
Yagi
,
H.
,
Izamis
,
M. L.
,
Guzzardi
,
M. A.
,
Shulman
,
C.
,
Milwid
,
J.
,
Kobayashi
,
N.
,
Tilles
,
A.
,
Berthiaume
,
F.
,
Hertl
,
M.
,
Nahmias
,
Y.
,
Yarmush
,
M. L.
, and
Uygun
,
K.
,
2010
, “
Organ Reengineering Through Development of a Transplantable Recellularized Liver Graft Using Decellularized Liver Matrix
,”
Nat. Med.
,
16
(
7
), pp.
814
820
.
4.
Zhang
,
Z.
, and
Michniak-Kohn
,
B. B.
,
2012
, “
Tissue Engineered Human Skin Equivalents
,”
Pharmaceutics
,
4
(
1
), pp.
26
41
.
5.
Keeney
,
M.
,
Lai
,
J. H.
, and
Yang
,
F.
,
2011
, “
Recent Progress in Cartilage Tissue Engineering
,”
Curr. Opin. Biotechnol.
,
22
(
5
), pp.
734
740
.
6.
Kock
,
L.
,
Van Donkelaar
,
C. C.
, and
Ito
,
K.
,
2012
, “
Tissue Engineering of Functional Articular Cartilage: The Current Status
,”
Cell Tissue Res.
,
347
(
3
), pp.
613
627
.
7.
Niklason
,
L. E.
,
Gao
,
J.
,
Abbott
,
W. M.
,
Hirschi
,
K. K.
,
Houser
,
S.
,
Marini
,
R.
, and
Langer
,
R.
,
1999
, “
Functional Arteries Grown in vitro
,”
Science
,
284
(
5413
), pp.
489
493
.
8.
Nemeno-Guanzon
,
J. G.
,
Lee
,
S.
,
Berg
,
J. R.
,
Jo
,
Y. H.
,
Yeo
,
J. E.
,
Nam
,
B. M.
,
Koh
,
Y. G.
, and
Lee
,
J. I.
,
2012
, “
Trends in Tissue Engineering for Blood Vessels
,”
J. Biomed. Biotechnol.
,
2012
(
2012
), p.
956345
.
9.
Atala
,
A.
,
Bauer
,
S. B.
,
Soker
,
S.
,
Yoo
,
J. J.
, and
Retik
,
A. B.
,
2006
, “
Tissue-Engineered Autologous Bladders for Patients Needing Cystoplasty
,”
Lancet
,
367
(
9518
), pp.
1241
1246
.
10.
Jednak
,
R.
,
2014
, “
The Evolution of Bladder Augmentation: From Creating a Reservoir to Reconstituting an Organ
,”
Front. Pediatr.
,
2
(
10
).
11.
Mironov
,
V.
,
Visconti
,
R. P.
,
Kasyanov
,
V.
,
Forgacs
,
G.
,
Drake
,
C. J.
, and
Markwald
,
R. R.
,
2009
, “
Organ Printing: Tissue Spheroids as Building Blocks
,”
Biomaterials
,
30
(
12
), pp.
2164
2174
.
12.
Nichol
,
J. W.
, and
Khademhosseini
,
A.
,
2009
, “
Modular Tissue Engineering: Engineering Biological Tissues from the Bottom Up
,”
Soft Matter
,
5
(
7
), pp.
1312
1319
.
13.
Sakai
,
Y.
,
Huang
,
H.
,
Hanada
,
S.
, and
Niino
,
T.
,
2010
, “
Toward Engineering of Vascularized Three-Dimensional Liver Tissue Equivalents Possessing a Clinically Significant Mass
,”
Biochem. Eng. J.
,
48
(
3
), pp.
348
361
.
14.
Wang
,
X.
,
Yan
,
Y.
, and
Zhang
,
R.
,
2010
, “
Recent Trends and Challenges in Complex Organ Manufacturing
,”
Tissue Eng. Part B Rev.
,
16
(
2
), pp.
189
197
.
15.
Guillotin
,
B.
, and
Guillemot
,
F.
,
2011
, “
Cell Patterning Technologies for Organotypic Tissue Fabrication
,”
Trends Biotechnol.
,
29
(
4
), pp.
183
190
.
16.
L'Heureux
,
N.
,
Dusserre
,
N.
,
Konig
,
G.
,
Victor
,
B.
,
Keire
,
P.
,
Wight
,
T. N.
,
Chronos
,
N. A. F.
,
Kyles
,
A. E.
,
Gregory
,
C. R.
,
Hoyt
,
G.
,
Robbins
,
R. C.
, and
Mcallister
,
T. N.
,
2006
, “
Human Tissue-Engineered Blood Vessels for Adult Arterial Revascularization
,”
Nat. Med.
,
12
(
3
), pp.
361
365
.
17.
Hannachi
,
I. E.
,
Itoga
,
K.
,
Kumashiro
,
Y.
,
Kobayashi
,
J.
,
Yamato
,
M.
, and
Okano
,
T.
,
2009
, “
Fabrication of Transferable Micropatterned-Co-Cultured Cell Sheets With Microcontact Printing
,”
Biomaterials
,
30
(
29
), pp.
5427
5432
.
18.
Williams
,
C.
,
Xie
,
A. W.
,
Yamato
,
M.
,
Okano
,
T.
, and
Wong
,
J. Y.
,
2011
, “
Stacking of Aligned Cell Sheets for Layer-by-Layer Control of Complex Tissue Structure
,”
Biomaterials
,
32
(
24
), pp.
5625
5632
.
19.
Lin
,
J. B.
,
Isenberg
,
B. C.
,
Shen
,
Y.
,
Schorsch
,
K.
,
Sazonova
,
O. V.
, and
Wong
,
J. Y.
,
2012
, “
Thermo-Responsive Poly(N-Isopropylacrylamide) Grafted onto Microtextured Poly(Dimethylsiloxane) for Aligned Cell Sheet Engineering
,”
Colloids Surf., B Biointerfaces
,
99
, pp.
108
115
.
20.
Du
,
Y. A.
,
Lo
,
E.
,
Ali
,
S.
, and
Khademhosseini
,
A.
,
2008
, “
Directed Assembly of Cell-Laden Microgels for Fabrication of 3d Tissue Constructs
,”
Proc. Natl. Acad. Sci. U.S.A.
,
105
(
28
), pp.
9522
9527
.
21.
Du
,
Y.
,
Lo
,
E.
,
Vidula
,
M. K.
,
Khabiry
,
M.
, and
Khademhosseini
,
A.
,
2008
, “
Method of Bottom-up Directed Assembly of Cell-Laden Microgels
,”
Cell. Mol. Bioeng.
,
1
(
2-3
), pp.
157
162
.
22.
Du
,
Y. A.
,
Ghodousi
,
M.
,
Lo
,
E.
,
Vidula
,
M. K.
,
Emiroglu
,
O.
, and
Khademhosseini
,
A.
,
2010
, “
Surface-Directed Assembly of Cell-Laden Microgels
,”
Biotechnol. Bioeng.
,
105
(
3
), pp.
655
662
.
23.
Zamanian
,
B.
,
Masaeli
,
M.
,
Nichol
,
J. W.
,
Khabiry
,
M.
,
Hancock
,
M. J.
,
Bae
,
H.
, and
Khademhosseini
,
A.
,
2010
, “
Interface-Directed Self-Assembly of Cell-Laden Microgels
,”
Small
,
6
(
8
), pp.
937
944
.
24.
Yanagawa
,
F.
,
Kaji
,
H.
,
Jang
,
Y. H.
,
Bae
,
H.
,
Du
,
Y. A.
,
Fukuda
,
J.
,
Qi
,
H.
, and
Khademhosseini
,
A.
,
2011
, “
Directed Assembly of Cell-Laden Microgels for Building Porous Three-Dimensional Tissue Constructs
,”
J. Biomed. Mater. Res. A
,
97A
(
1
), pp.
93
102
.
25.
Fedorovich
,
N. E.
,
Swennen
,
I.
,
Girones
,
J.
,
Moroni
,
L.
,
Van Blitterswijk
,
C. A.
,
Schacht
,
E.
,
Alblas
,
J.
, and
Dhert
,
W. J. A.
,
2009
, “
Evaluation of Photocrosslinked Lutrol Hydrogel for Tissue Printing Applications
,”
Biomacromolecules
,
10
(
7
), pp.
1689
1696
.
26.
Iwami
,
K.
,
Noda
,
T.
,
Ishida
,
K.
,
Morishima
,
K.
,
Nakamura
,
M.
, and
Umeda
,
N.
,
2010
, “
Bio Rapid Prototyping by Extruding/Aspirating/Refilling Thermoreversible Hydrogel
,”
Biofabrication
,
2
(
1
), p.
014108
.
27.
Skardal
,
A.
,
Zhang
,
J. X.
, and
Prestwich
,
G. D.
,
2010
, “
Bioprinting Vessel-Like Constructs Using Hyaluronan Hydrogels Crosslinked with Tetrahedral Polyethylene Glycol Tetracrylates
,”
Biomaterials
,
31
(
24
), pp.
6173
6181
.
28.
Song
,
S. J.
,
Choi
,
J.
,
Park
,
Y. D.
,
Hong
,
S.
,
Lee
,
J. J.
,
Ahn
,
C. B.
,
Choi
,
H.
, and
Sun
,
K.
,
2011
, “
Sodium Alginate Hydrogel-Based Bioprinting Using a Novel Multinozzle Bioprinting System
,”
Artif. Organs
,
35
(
11
), pp.
1132
1136
.
29.
Chang
,
R.
,
Nam
,
J.
, and
Sun
,
W.
,
2008
, “
Effects of Dispensing Pressure and Nozzle Diameter on Cell Survival from Solid Freeform Fabrication-Based Direct Cell Writing
,”
Tissue Eng. Part A
,
14
(
1
), pp.
41
48
.
30.
Tirella
,
A.
,
Vozzi
,
F.
,
Vozzi
,
G.
, and
Ahluwalia
,
A.
,
2011
, “
Pam2 (Piston Assisted Microsyringe): A New Rapid Prototyping Technique for Biofabrication of Cell Incorporated Scaffolds
,”
Tissue Eng. Part C Methods
,
17
(
2
), pp.
229
237
.
31.
Xu
,
C.
,
Chai
,
W.
,
Huang
,
Y.
, and
Markwald
,
R. R.
,
2012
, “
Scaffold-Free Inkjet Printing of Three-Dimensional Zigzag Cellular Tubes
,”
Biotechnol. Bioeng.
,
109
(
12
), pp.
3152
3160
.
32.
Pataky
,
K.
,
Braschler
,
T.
,
Negro
,
A.
,
Renaud
,
P.
,
Lutolf
,
M. P.
, and
Brugger
,
J.
,
2012
, “
Microdrop Printing of Hydrogel Bioinks into 3d Tissue-Like Geometries
,”
Adv. Mater.
,
24
(
3
), pp.
391
396
.
33.
Yu
,
Y.
,
Zhang
,
Y.
,
Martin
,
J. A.
, and
Ozbolat
,
I. T.
,
2013
, “
Evaluation of Cell Viability and Functionality in Vessel-Like Bioprintable Cell-Laden Tubular Channels
,”
ASME J. Biomech. Eng.
,
135
(
9
), p.
91011
.
34.
Cohen
,
D. L.
,
Malone
,
E.
,
Lipson
,
H.
, and
Bonassar
,
L. J.
,
2006
, “
Direct Freeform Fabrication of Seeded Hydrogels in Arbitrary Geometries
,”
Tissue Eng.
,
12
(
5
), pp.
1325
1335
.
35.
Cohen
,
D. L.
,
Lo
,
W.
,
Tsavaris
,
A.
,
Peng
,
D.
,
Lipson
,
H.
, and
Bonassar
,
L. J.
,
2011
, “
Increased Mixing Improves Hydrogel Homogeneity and Quality of Three-Dimensional Printed Constructs
,”
Tissue Eng. Part C, Methods
,
17
(
2
), pp.
239
248
.
36.
Atala
,
A.
,
Kasper
,
F. K.
, and
Mikos
,
A. G.
,
2012
, “
Engineering Complex Tissues
,”
Sci. Transl. Med.
,
4
(
160
), p.
160rv12
.
37.
Derby
,
B.
,
2012
, “
Printing and Prototyping of Tissues and Scaffolds
,”
Science
,
338
(
6109
), pp.
921
926
.
38.
Ozbolat
,
I. T.
,
2015
, “
Bioprinting Scale-up Tissue and Organ Constructs for Transplantation
,”
Trends Biotechnol.
,
33
(
7
), pp.
395
400
.
39.
Ozbolat
,
I. T.
,
Chen
,
H.
, and
Yu
,
Y.
,
2014
, “
Development of ‘Multi-Arm Bioprinter’ for Hybrid Biofabricationof Tissue Engineering Constructs
,”
Robot Comput. Integr. Manuf.
,
30
(
3
), pp.
295
304
.
40.
Kolesky
,
D. B.
,
Truby
,
R. L.
,
Gladman
,
A. S.
,
Busbee
,
T. A.
,
Homan
,
K. A.
, and
Lewis
,
J. A.
,
2014
, “
3D Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue Constructs
,”
Adv. Mater.
,
26
(
19
), pp.
3124
3130
.
41.
Herring
,
M.
,
Gardner
,
A.
, and
Glover
,
J.
,
1978
, “
A Single-Staged Technique for Seeding Vascular Grafts with Autogenous Endothelium
,”
Surgery
,
84
(
4
), pp.
498
504
.
42.
L'Heureux
,
N.
,
Paquet
,
S.
,
Labbe
,
R.
,
Germain
,
L.
, and
Auger
,
F. A.
,
1998
, “
A Completely Biological Tissue-Engineered Human Blood Vessel
,”
FASEB J.
,
12
(
1
), pp.
47
56
.
43.
Dahl
,
S. L.
,
Koh
,
J.
,
Prabhakar
,
V.
, and
Niklason
,
L. E.
,
2003
, “
Decellularized Native and Engineered Arterial Scaffolds for Transplantation
,”
Cell Transplant
,
12
(
6
), pp.
659
666
.
44.
Tamura
,
N.
,
Nakamura
,
T.
,
Terai
,
H.
,
Iwakura
,
A.
,
Nomura
,
S.
,
Shimizu
,
Y.
, and
Komeda
,
M.
,
2003
, “
A New Acellular Vascular Prosthesis as a Scaffold for Host Tissue Regeneration
,”
Int. J. Artif. Organs
,
26
(
9
), pp.
783
792
.
45.
Uchimura
,
E.
,
Sawa
,
Y.
,
Taketani
,
S.
,
Yamanaka
,
Y.
,
Hara
,
M.
,
Matsuda
,
H.
, and
Miyake
,
J.
,
2003
, “
Novel Method of Preparing Acellular Cardiovascular Grafts by Decellularization With Poly(Ethylene Glycol)
,”
J. Biomed. Mater. Res. A
,
67A
(
3
), pp.
834
837
.
46.
Nakamura
,
M.
,
Iwanaga
,
S.
,
Henmi
,
C.
,
Arai
,
K.
, and
Nishiyama
,
Y.
,
2010
, “
Biomatrices and Biomaterials for Future Developments of Bioprinting and Biofabrication
,”
Biofabrication
,
2
(
1
), p.
014110
.
47.
Wu
,
W.
,
Deconinck
,
A.
, and
Lewis
,
J. A.
,
2011
, “
Omnidirectional Printing of 3D Microvascular Networks
,”
Adv. Mater.
,
23
(
24
), pp.
H178
H183
.
48.
Norotte
,
C.
,
Marga
,
F. S.
,
Niklason
,
L. E.
, and
Forgacs
,
G.
,
2009
, “
Scaffold-Free Vascular Tissue Engineering Using Bioprinting
,”
Biomaterials
,
30
(
30
), pp.
5910
5917
.
49.
Yan
,
Y.
,
Wang
,
X.
,
Pan
,
Y.
,
Liu
,
H.
,
Cheng
,
J.
,
Xiong
,
Z.
,
Lin
,
F.
,
Wu
,
R.
,
Zhang
,
R.
, and
Lu
,
Q.
,
2005
, “
Fabrication of Viable Tissue-Engineered Constructs With 3D Cell-Assembly Technique
,”
Biomaterials
,
26
(
29
), pp.
5864
5871
.
50.
Smith
,
C. M.
,
Christian
,
J. J.
,
Warren
,
W. L.
, and
Williams
,
S. K.
,
2007
, “
Characterizing Environmental Factors That Impact the Viability of Tissue-Engineered Constructs Fabricated by a Direct-Write Bioassembly Tool
,”
Tissue Eng.
,
13
(
2
), pp.
373
383
.
51.
Smith
,
C. M.
,
Stone
,
A. L.
,
Parkhill
,
R. L.
,
Stewart
,
R. L.
,
Simpkins
,
M. W.
,
Kachurin
,
A. M.
,
Warren
,
W. L.
, and
Williams
,
S. K.
,
2004
, “
Three-Dimensional Bioassembly Tool for Generating Viable Tissue-Engineered Constructs
,”
Tissue Eng.
,
10
(
9-10
), pp.
1566
1576
.
You do not currently have access to this content.